Cellular strategies for regulating DNA supercoiling: a single-molecule perspective.

Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal.

Microbial Degradation Rates of Natural Bitumen.

Microorganisms are present in nearly every oil or bitumen sample originating from temperate reservoirs. Nevertheless, it is very difficult to obtain reliable estimates about microbial processes taking place in deep reservoirs, since metabolic rates are rather low and differ strongly during artificially cultivation. Here, we demonstrate the importance and impact of microorganisms entrapped in microscale water droplets for the overall biodegradation process in bitumen. To this end, we measured degradation rates of heavily biodegraded bitumen from the Pitch Lake (Trinidad and Tobago) using the novel technique of reverse stable isotope labeling, allowing precise measurements of comparatively low mineralization rates in the ng range in microcosms under close to natural conditions. Freshly taken bitumen samples were overlain with artificial brackish water and incubated for 945 days. Additionally, three-dimensional distribution of water droplets in bitumen was studied with computed tomography, revealing a water bitumen interface of 1134 cm2 per liter bitumen, resulting in an average mineralization rate of 9.4-38.6 mmol CO2 per liter bitumen and year. Furthermore, a stable and biofilm-forming microbial community established on the bitumen itself, mainly composed of fermenting and sulfate-reducing bacteria. Our results suggest that small water inclusions inside the bitumen substantially increase the bitumen-water interface and might have a major impact on the overall oil degradation process.

You are how you eat: differences in trophic position of two parasite species infecting a single host according to stable isotopes.

Parasitism is commonly recognised as a consumer strategy, although, the interaction of parasites in communities and ecosystems are generally poorly understood. As parasites are integral parts of food webs, analysis of the trophic interactions between parasites and hosts was assessed through comparison of stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N). Largemouth yellowfish (Labeobarbus kimberleyensis) infected with the Asian tapeworm (Schyzocotyle acheilognathi) were collected from the Vaal Dam. Signatures of δ13C and δ15N were assessed in host muscle and liver tissue, and cestodes using an elemental analyser coupled with an isotope ratio-mass spectrometer (EA-IRMS). Hosts were enriched by 4.1‰ in the heavy nitrogen isotope with respect to the S. acheilognathi and therefore occupy a higher trophic position than the parasite. Comparison of δ13C indicates that dietary sources of carbon in cestodes are derived from the host liver. Comparison of stable isotope signatures between Paradiplozoon ichthyoxanthon (another common parasite of the Largemouth yellowfish in the Vaal River) and S. acheilognathi showed that the monogenean was enriched by 5.3‰ in 15N which accounts for a difference of almost two trophic positions. Isotope differences in the host-parasite system considered indicate that differences can be related to the mode of nutrient acquisition employed by host and parasites. Cestodes, being depleted in both 13C and 15N relative to the host and monogenean (P. ichthyoxanthon), indicate that S. acheilognathi assimilates nutrients derived from the host metabolism which are released from the liver.

New Concepts for the Determination of Oxidation Efficiencies in Liquid Chromatography-Isotope Ratio Mass Spectrometry.

In liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS), analytes are separated on an LC system and consecutively oxidized to CO2, which is required for the determination of compound-specific carbon isotope ratios. Oxidation is performed in an online reactor by sulfate radicals. Reaction conditions in the interface depend on the flow conditions determined by the LC method and the flow rates and concentrations of oxidation agent and phosphoric acid added in the interface. To determine accurate isotope ratios, a quantitative conversion of the carbon contained in the analyte to the CO2 measurement gas is a prerequisite. Oxidation efficiencies are not commonly evaluated during method development, although certain analytes are known to be difficult to be oxidized by sulfate radicals. For the assessment of the oxidation efficiency of the LC-IRMS system, three different approaches were evaluated. (1) Residual organic carbon in the eluent stream of the interface was determined to calculate oxidation yields depending on the initial analyte concentration. (2) The IRMS response was calibrated to an inorganic carbon reference material to determine oxidation efficiencies with the help of the IRMS as a detector. (3) The oxidation temperature was deliberately reduced while monitoring the δ13C and signal intensity. The common assumption that a linear relation of IRMS signal to analyte concentration is an indicator for complete oxidation in LC-IRMS could be disproved. All three approaches can be applied for future method development in LC-IRMS, monitoring of existing flow injection applications, as well as for verification of complete oxidation in established LC-IRMS methods.

Degradation of the Polymeric Brominated Flame Retardant "Polymeric FR" by Heat and UV Exposure.

Monomeric brominated flame retardants often pose risks to the environment. The new group of polymeric flame retardants is claimed to be a safer alternative due to their high molecular weight and persistence by design. Within this publication, the degradation of a commercially widely applied example of this group-the polymer "Polymeric FR"-was studied during UV irradiation and long-term exposure to heat (60 °C) for up to 36 weeks. Both treatments led to a variety of degradation products, which might have potentially adverse environmental effects and an increased mobility compared to the mother polymer. Besides identifying some of the possible degradation products (including for instance 2,4,6-tribromo-3-hydroxybenzoic acid), the degradation via UV irradiation, which yields 75 different degradation products, and via heat, which led to significantly less products, was compared. In addition, further parameters like TOC and the concentration of free bromine were studied and it was demonstrated that the used type of water (distilled, reconstituted, and rainwater) does not influence the outcome of the degradation experiments.

Bacterial predator-prey dynamics in microscale patchy landscapes.

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales. We also found that while the prey population in the continuous landscape was almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions.

Antitumour drugs impede DNA uncoiling by topoisomerase I.

Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks. Here we use single-molecule nanomanipulation to monitor the dynamics of human topoisomerase I in the presence of topotecan. This allowed us to detect the binding and unbinding of an individual topotecan molecule in real time and to quantify the drug-induced trapping of topoisomerase on DNA. Unexpectedly, our findings also show that topotecan significantly hinders topoisomerase-mediated DNA uncoiling, with a more pronounced effect on the removal of positive (overwound) versus negative supercoils. In vivo experiments in the budding yeast verified the resulting prediction that positive supercoils would accumulate during transcription and replication as a consequence of camptothecin poisoning of topoisomerase I. Positive supercoils, however, were not induced by drug treatment of cells expressing a catalytically active, camptothecin-resistant topoisomerase I mutant. This combination of single-molecule and in vivo data suggests a cytotoxic mechanism for camptothecins, in which the accumulation of positive supercoils ahead of the replication machinery induces potentially lethal DNA lesions.

Biomonitoring of polycyclic aromatic hydrocarbons in firefighters at fire training facilities and in employees at respiratory protection and hose workshops.

Introduction: Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic to humans and are formed by incomplete combustion. PAHs are always present during firefighting operations, and fire department members can be exposed to them in the workplace. Methods: In this study, we analyzed 1-hydroxypyrene (1-OHP) in 36 urine samples from nine firefighters, collected before and after fire training sessions, and 32 urine samples from eight employees at respiratory protection and hose workshops. To assess breakthrough PAH exposure through personal protective equipment and potential dermal uptake, some of the workshop employees wore cotton garments under their regular workwear. Cotton samples were then examined for the presence of 17 semi-volatile and low-volatility PAHs. Results: After firefighting exercises, we observed approximately a fivefold increase in mean 1-OHP concentrations in samples from firefighters, from 0.24 µg/L to 1.17 µg/L (maximum: 5.31 µg/L). In contrast, 1-OHP levels in workshop employees were found to be low, with the majority of urine samples yielding concentrations below the limit of quantification (LOQ: 0.05 µg/L, maximum: 0.11 µg/L). Similarly, low PAH levels were found on the workshop employees' cotton undergarments, with maximum concentrations of 250 and 205 ng/g for pyrene and benzo[a]pyrene, respectively. Discussion: In conclusion, significant increases in 1-OHP in urine were observed in firefighters after training sessions, whereas work-related exposure remained low among workshop employees.

Genetic diversity in terrestrial subsurface ecosystems impacted by geological degassing.

Earth's mantle releases 38.7 ± 2.9 Tg/yr CO2 along with other reduced and oxidized gases to the atmosphere shaping microbial metabolism at volcanic sites across the globe, yet little is known about its impact on microbial life under non-thermal conditions. Here, we perform comparative metagenomics coupled to geochemical measurements of deep subsurface fluids from a cold-water geyser driven by mantle degassing. Key organisms belonging to uncultivated Candidatus Altiarchaeum show a global biogeographic pattern and site-specific adaptations shaped by gene loss and inter-kingdom horizontal gene transfer. Comparison of the geyser community to 16 other publicly available deep subsurface sites demonstrate a conservation of chemolithoautotrophic metabolism across sites. In silico replication measures suggest a linear relationship of bacterial replication with ecosystems depth with the exception of impacted sites, which show near surface characteristics. Our results suggest that subsurface ecosystems affected by geological degassing are hotspots for microbial life in the deep biosphere.

Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB.

Topoisomerases relieve the torsional strain in DNA that is built up during replication and transcription. They are vital for cell proliferation and are a target for poisoning by anti-cancer drugs. Type IB topoisomerase (TopIB) forms a protein clamp around the DNA duplex and creates a transient nick that permits removal of supercoils. Using real-time single-molecule observation, we show that TopIB releases supercoils by a swivel mechanism that involves friction between the rotating DNA and the enzyme cavity: that is, the DNA does not freely rotate. Unlike a nicking enzyme, TopIB does not release all the supercoils at once, but it typically does so in multiple steps. The number of supercoils removed per step follows an exponential distribution. The enzyme is found to be torque-sensitive, as the mean number of supercoils per step increases with the torque stored in the DNA. We propose a model for topoisomerization in which the torque drives the DNA rotation over a rugged periodic energy landscape in which the topoisomerase has a small but quantifiable probability to religate the DNA once per turn.

Dynamics of phosphodiester synthesis by DNA ligase.

Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for approximately 10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligase-AppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s(-1)) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.

Eye fluke infection changes diet composition in juvenile European perch (Perca fluviatilis).

Intraspecific diet specialization, usually driven by resource availability, competition and predation, is common in natural populations. However, the role of parasites on diet specialization of their hosts has rarely been studied. Eye flukes can impair vision ability of their hosts and have been associated with alterations of fish feeding behavior. Here it was assessed whether European perch (Perca fluviatilis) alter their diet composition as a consequence of infection with eye flukes. Young-of-the-year (YOY) perch from temperate Lake Müggelsee (Berlin, Germany) were sampled in two years, eye flukes counted and fish diet was evaluated using both stomach content and stable isotope analyses. Perch diet was dominated by zooplankton and benthic macroinvertebrates. Both methods indicated that with increasing eye fluke infection intensity fish had a more selective diet, feeding mainly on the benthic macroinvertebrate Dikerogammarus villosus, while less intensively infected fish appeared to be generalist feeders showing no preference for any particular prey type. Our results show that infection with eye flukes can indirectly affect interaction of the host with lower trophic levels by altering the diet composition and highlight the underestimated role of parasites in food web studies.

Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence.

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB-DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drug-bound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB-drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB-drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequence-dependent dynamics need not to be taken into account in efforts to develop novel camptothecins.

Stable isotope analysis spills the beans about spatial variance in trophic structure in a fish host - parasite system from the Vaal River System, South Africa.

Stable isotope analysis offers a unique tool for comparing trophic interactions and food web architecture in ecosystems based on analysis of stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) in organisms. Clarias gariepinus were collected from six sites along the Vaal River, South Africa and were assessed for ectoparasites and endoparasites. Lamproglena clariae (Copepoda), Tetracampos ciliotheca and Proteocephalus glanduligerus (Cestoda), and larval Contracaecum sp. (Nematoda) were collected from the gills, intestine and mesenteries, respectively. Signatures of δ13C and δ15N were analysed in host muscle tissue and parasites using bulk stable isotope analysis. Variable stable isotope enrichment between parasites and host were observed; L. clariae and the host shared similar δ15N signatures and endoparasites being depleted in δ13C and δ15N relative to the host. Differences in stable isotope enrichment between parasites could be related to the feeding strategy of each parasite species collected. Geographic and spatial differences in enrichment of stable isotopes observed in hosts were mirrored by parasites. As parasites rely on a single host for meeting their nutritional demands, stable isotope variability in parasites relates to the dietary differences of host organisms and therefore variations in baseline stable isotope signatures of food items consumed by hosts.

Origin of Xylitol in Chewing Gum: A Compound-Specific Isotope Technique for the Differentiation of Corn- and Wood-Based Xylitol by LC-IRMS.

The sugar replacement compound xylitol has gained increasing attention because of its use in many commercial food products, dental-hygiene articles, and pharmaceuticals. It can be classified by the origin of the raw material used for its production. The traditional "birch xylitol" is considered a premium product, in contrast to xylitol produced from agriculture byproducts such as corn husks or sugar-cane straw. Bulk stable-isotope analysis (BSIA) and compound-specific stable-isotope analysis (CSIA) by liquid-chromatography isotope-ratio mass spectrometry (LC-IRMS) of chewing-gum extracts were used to determine the δ13C isotope signatures for xylitol. These were applied to elucidate the original plant type the xylitol was produced from on the basis of differences in isotope-fractionation processes of photosynthetic CO2 fixation. For the LC-IRMS analysis, an organic-solvent-free extraction protocol and HPLC method for the separation of xylitol from different artificial sweeteners and sugar-replacement compounds was successfully developed and applied to the analysis of 21 samples of chewing gum, from which 18 could be clearly related to the raw-material plant class.

Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion.

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB-DNA complexes has suggested that the enzyme promotes long-range DNA-DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg2+, Ca2+ or Mn2+, suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge.

Subjective Cognitive Complaints in Parkinson Disease Without Dementia: A Preliminary Study.

Little is known about the subjective cognitive complaints of individuals with Parkinson disease (PD). Such complaints have become a topic of interest recently as they play a role in the diagnosis of neurocognitive disorders. The aim of this preliminary study was to determine whether a sample of nondemented individuals with PD reported significantly more difficulties with multiple elements of cognition than a control sample and to assess the relation between their ratings and demographics, motor symptom severity, neuropsychological test performance, and measures of depression and anxiety. Forty nondemented individuals with PD and 27 healthy individuals completed a questionnaire assessing everyday cognitive difficulties. Independent t tests indicated that individuals with PD reported significantly more cognitive complaints in general and in specific tasks involving complex attention, executive function, processing speed, and verbal fluency but not memory. Questionnaire ratings significantly correlated with measures assessing anxiety, verbal memory, processing speed, and verbal fluency. Results suggest that it is important to ask individuals with PD about cognitive complaints across several cognitive domains and also inquire about symptoms of anxiety, which may be related to their self-reported cognitive difficulties.

Environmental assessment of digestate treatment technologies using LCA methodology.

The production of biogas from energy crops, organic waste and manure has augmented considerably the amounts of digestate available in Flanders. This has pushed authorities to steadily introduce legislative changes to promote its use as a fertilising agent. There is limited arable land in Flanders, which entails that digestate has to compete with animal manure to be spread. This forces many anaerobic digestion plants to further treat digestate in such a way that it can either be exported or the nitrogen be removed. Nevertheless, the environmental impact of these treatment options is still widely unknown, as well as the influence of these impacts on the sustainability of Flemish anaerobic digestion plants in comparison to other regions where spreading of raw digestate is allowed. Despite important economic aspects that must be considered, the use of Life Cycle Assessment (LCA) is suggested in this study to identify the environmental impacts of spreading digestate directly as compared to four different treatment technologies. Results suggest relevant environmental gains when the digestate mix is treated using the examined conversion technologies prior to spreading, although important trade-offs between impact categories were observed and discussed. The promising results of digestate conversion technologies suggest that further LCA analyses should be performed to delve into, for instance, the appropriateness to shift to nutrient recovery technologies rather than digestate conversion treatments.